Electrophoresis is a well known technique for separating complex organic molecules. In recent years, electrophoresis has been used extensively in the separation, isolation and analysis of proteins. Electrophoresis has the advantage of being able to separate a large number of proteins from a very small sample.
In the typical electrophoretic separation of macromolecules, such as nucleic acids and proteins, an electric potential is applied to opposite ends of a substrate that is in the presence of an electrolyte. The electric current causes the macromolecules to migrate through the medium along the substrate to a location determined by the size and charge of the molecules, the substrate retarding movement and the current. Typically, the electrolyte is an inorganic salt in an aqueous medium.
One type of electrophoresis process uses a capillary electrophoresis device. This device includes one or more capillary tubes or channels containing the electrolyte solution. An electric field is created in the electrolyte solution between the ends of the capillary to cause the macromolecules to migrate along the length of the capillary. The velocity of the sample component through the capillary tube is a function of the electric field, the movement of the carrier liquid and the electric mobility of the sample component. One example of a capillary electrophoresis device is disclosed in U.S. Pat. No. 6,159,353 to West et al.
Capillary electrophoresis devices are typically an enclosed capillary tube. The capillary tubes generally have a length of about 1 to 2 meters. The voltage of the electric current applied to the electrolyte solution can range from about 5 to 50 kilovolts. The separation of the constituents of the sample is the result of the differential migration through the gel or capillary tube due to the charge to size ratio or by chemical interactions between the samples and a stationary phase in the capillary or gel. The efficiency of the separation is enhanced by reducing the capillary diameter and the thickness of the gel.
Other types of electrophoresis separation processes use a gel supported in a tube. This electrophoresis separation is generally referred to as a first dimension separation when combined with an additional subsequent separation. The tube contains an electrophoresis gel where each end of the tube is immersed in an aqueous solution containing a buffer or other electrolyte. The gel is commonly an acrylamide gel that contains large amounts of water. A test sample is applied to one end of the tube and an electric potential is applied between opposite ends of the tube. The components of the test sample migrate along the length of the tube a distance according to the charge and mass of the component. When electrophoresis is performed in the presence of ampholytes, the components may focus based on charge alone.
Another electrophoresis process employs a gel slab or a thin planar gel. The gels generally have a length of about 5 to 100 cm. When preceded by a first dimensional separation, it is commonly referred to as second dimension electrophoresis. The gel slab is typically a sheet having a thickness of about 1 mm to 3 mm and is supported between two plates. Typically, the supports are two sheets of glass with spacers between the sheets to maintain a uniform spacing. Test samples can be applied to one end of the gel, in a discrete location or a tubular shaped gel containing separated molecules obtained from a first dimension electrophoresis separation process that has been separated from the tube. In other forms, liquid samples containing molecules to be separated are placed along a side edge of the gel slab or in wells. The ends of the gel slab are immersed in a buffer solution and an electric potential is applied to opposite ends to cause the molecules of the sample to migrate through the gel.
Electrophoresis is a particularly suitable technique for the separation and isolation of many proteins. The prior electrophoresis processes and devices use an aqueous system containing an electrolyte and where the gel material is formed by contacting the polymer and the aqueous system. The presently available electrophoresis systems are limited by the ability to solubilize the proteins in an aqueous medium using buffers, salts, denaturants, detergents or other solubilizing agents. Many proteins that are highly hydrophobic cannot be solubilized in aqueous systems, and thus, cannot be subjected to electrophoresis separation using conventional electrophoresis processes. If one uses a solubilizing agent which imparts a charge to the molecule, it may solubilize but loose its specific charge to size ratio and the basis for its separation ability during electrophoresis. Certain proteins under controlled conditions can be solubilized in organic solvents. However, the organic solvents are not compatible with the electrophoresis gels or the aqueous systems used to form the gel. The organic solvents are also not electrically conductive to be amenable to electrophoresis.
Efforts have been made to solubilize or suspend hydrophobic proteins in aqueous solutions for various processes. For example, Wissing et al. in “Enrichment of Hydrophobic Proteins via Triton X-114 Phase Portioning and Hydroxyapatite Column Chromatography for Mass Spectrometry”, Electrophoresis, 2000, 21, 2589-2593 discloses the separation of hydrophobic membrane proteins. The process discloses forming an aqueous solution of the nonionic detergent Triton X-114, which at 20° C. separates into a detergent depleted phase and a detergent enriched phase. The detergent enriched phase is disclosed as containing the hydrophobic proteins. The proteins were separated by column chromatography, followed by electrophoresis using IPG strips. The gels were then stained, the spots excised and the peptides extracted for analysis by MALDI-TOF.
Low temperature molten salts are a class of salt compounds and compositions commonly referred to as ionic liquids. Ionic liquids are generally liquid at room temperature and are made up of organic cations and anions. Ionic liquids have been proposed for use as solvents, for catalysts, organic synthesis and non-aqueous batteries. Examples of electrochemical cells that include ionic liquids are disclosed in U.S. Pat. No. 5,552,241 to Manantov et al. and U.S. Pat. No. 6,326,104 to Caja et al.
Accordingly, a continuing need exists in the industry for improved processes for solubilizing hydrophobic compounds and the electrophoretic separation of these molecules.